Diabetes - a major health problem for veterans - is due to loss of beta-cell mass and function. Since decreased beta-cell mass underlies the development and progression of both Type 1 and Type 2 diabetes (DM), understanding the regulation of beta-cell mass is imperative for both prevention and treatment. Although the mechanisms regulating beta-cell mass are poorly understood, glial cell line-derived neurotrophic factor (GDNF) may play an important role. We have previously shown that GDNF transgenic mice (GDNF-tg, engineered to over-express GDNF in glia) have increased beta cell mass and resist streptozotocin-induced hyperglycemia. In order to guide the use of GDNF, its receptor agonists, or its signal transduction targets as a basis for development of agents to promote beta-cell mass, we first need better understanding of mechanism of GDNF action on the beta-cell. Our hypothesis is that GDNF promotes increased beta-cell mass by promoting beta-cell differentiation and protects against diabetes by enhancing beta- cell mass, survival and regeneration. Further, GDNF improves beta-cell (islet) transplantation by enhancing beta-cell survival. In specific Aim 1 we will determine the role of Pdx-1 in GDNF-induced pancreatic beta-cell differentiation. Preliminary data demonstrates that GDNF-mediated differentiation of 2-TC-6 cells and HIT cells is reduced in the presence of Pdx-1 siRNA. Using in vitro models of beta-cell differentiation (2-TC-6 cells and HIT cells) in conjunction with specific siRNA, promoter assays and DNA binding assays, the necessity and sufficiency of Pdx-1 in modulating GDNF-induced beta-cell differentiation will be determined. In specific aim 2 we will determine the role of Sox-9 and Pdx-1 in GDNF induced beta-cell regeneration using in vitro (thapsigargin induce 2-TC-6 cell injury) in conjunction with RNAi and in vivo (streptozotocin treated GDFN-tg and WT mice) models. Preliminary data shows GDNF induced beta-cell proliferation is reduced in the presence of SOX9 siRNA. Using a co-culture model of neurons/glia on beta-cells we will determine the mechanism of interaction of enteric neurons and beta-cells. In Specific Aim 3 we will assess the effect of GDNF on murine and porcine islet post- transplantation survival. Preliminary data shows increased post transplantation survival of mouse islets pre-cultured with GDNF. Porcine islets can be used for human transplantation. We will pre-treat murine and porcine islets with vehicle or GDNF and perform intrahepatic portal vein transplantation in diabetic mice. The efect of GDNF and enteric neurons/glia on post transplantation survival of islets wil be assessed by evaluating beta cell mass and function. Impact on Veterans Health Care: Diabetes is highly prevalent in the Veteran population and the incidence is rising. Results from this study may help us identify new targets to improve beta cell mass to help in the prevention or treatment of diabetes. Identification of new therapies for diabetes will directly benefit the veteran population.